Anti-inflammatory peptides derived from C-reactive protein

ABSTRACT

A peptide corresponding to positions 62-71 of the sequence of human C-reactive protein (CRP) of the formula: Glu 62 -Ile-Leu-Ile-Phe-Ser-Lys-ASP-Ile 71  and modifications thereof obtained by substitution, deletion, or addition of amino acids, amidation of the C-terminal or acylation of the N-terminal, are capable of inhibiting in vitro the enzymatic activity of human Leukocyte Elastase (hLE) and/or of human Cathepsin G (hCG) and can be used for the treatment of chronic inflammation conditions such as rheumatoid arthritis, pulmonary emphysema, cystic fibrosis, bronchitis, asthma and acute respiratory distress syndrome.

CROSS REFERENCE TO RELATED APPLICATION

The present application is the national stage under 35 U.S.C. 371 ofPCT/IL98/00302, filed Jun. 29, 1998.

FIELD OF THE INVENTION

The present invention relates to synthetic peptides derived from theprimary sequence of the acute phase reactant C-reactive protein (CRP)(SEQ ID NO:1), more particularly to a peptide corresponding to positions62-71 of CRP and derivatives thereof, which peptides inhibit in vitrothe enzymatic activities of human leukocyte elastase (hLE) and/or humanleukocyte cathepsin G (hCG), two potent serine proteases associated withtissue damage occurring in the course of several chronic inflammatoryconditions. The invention further relates to anti-inflammatorypharmaceutical compositions comprising said CRP-derived peptides.

Abbreviations

The following abbreviations will be used throughout the specification:

CRP, C-reactive protein; hLE, human leukocyte elastase; hCG, humanleukocyte cathepsin G; MeOSuc-AAPV-NA,methoxysuccinyl-Ala-Ala-Pro-Val-nitroanilide (SEQ ID NO:2), Suc-AAPF-NA,succinyl-Ala-Ala-Pro-Phe-nitroanilide (SEQ ID NO:3).

BACKGROUND OF THE INVENTION

C-reactive protein (CRP) is a plasma protein classified as a major acutephase reactant due to its dramatic accumulation in the blood streamduring the inflammatory response. Within a relatively short period(24-48 hr) following tissue injury or certain traumatic events, the CRPblood concentration may rise 1000-fold over the normal level to as highas 1 mg/ml (Ballue and Kushner, 1992).

CRP consists of five identical sub-units that contain each 206 aminoacids bridged by a single disulfide bond and that aggregatenon-covalently into a cyclic pentamer termed pentraxin. The precisebiochemical function of CRP as a whole entity is still obscure. CRP wasshown to bind to specific receptors on human neutrophils (K_(d)˜5×10⁻⁸M), monocytes (K_(d)˜10⁻⁷ M), and other inflammatory-related cells invitro (Ballue and Kushner, 1992),

In the laboratories of the present inventors and their collaborators itwas found that following binding to neutrophils, CRP is subsequentlydegraded by a membrane-associated neutral serine protease, which hasbeen characterized (Shephard et al., 1992), and by lysosomal-derivedenzymes to yield various low molecular weight peptides. Several of thesepeptides were identified, synthesized, and shown to be potentanti-inflammatory agents inhibiting neutrophil phagocytosis,degranulation, and superoxide ion (O₂) generation (Shephard et al.,1990; Yavin et al., 1995). Superoxide ion is the parent compound ofseveral destructive mediators that are believed to play a central rolein inflammation-associated tissue injury (Ballue and Kushner, 1992).

The most prominent of the peptides disclosed by Shephard et al., 1990,and Yavin et al., 1995, were derived from within the primary sequence ofCRP (SEQ ID NO:1) as follows: Asp₇₀-Ile-Gly-Tyr-Ser₇₄,Lys₂₀₁-Pro-Gln-Leu-Trp-Pro₂₀₆, Leu₈₃-Phe-Glu-Val-Pro-Glu-Val-Thr₉₀,Val-₇₇-Gly-Gly-Ser-Glu-Ile₈₂ (Shephard et al., 1990) andAsn₁₆₀-Met-Trp-Asp-Phe-Val₁₆₅, Gln₂₀₃-Leu-Trp-Pro₂₀₆,Ser₁₈-Tyr-Val-Ser-Leu-Lys₂₃ (Yavin et al., 1995). These peptides wereshown by the authors to inhibit various neutrophilic functions,indicating that they may be capable of regulating superoxide ionproduction by neutrophils in vivo during the acute phase response aspart of a complex protective mechanism. However, as disclosed in the PCTPublication No. WO 97/28182 of the same applicants, several of thesepeptides lack hLE inhibitory capability.

Human leukocyte elastase (hLE) and human leukocyte cathepsin G (hCG) arethe two major potent neutral serine proteases found in the azurophilicgranules of neutrophils which are involved in the intracellulardigestion of proteins and play an important role in phagocytosis andhost defense against invading organisms. In the extracellularenvironment, hLE is capable of degrading various connective tissueproteins including highly cross-linked elastin whereas hCG is veryeffective in degrading proteoglycans and collagens and has been shown toaugment the elastolytic capability of hLE (Groutas, 1987).

The release of enzymes into the extracellular medium by activatedneutrophils is normally controlled by several potent inhibitors. Themost specific natural inhibitors, α₁-protease inhibitor (α₁-PI) andα-antichymotrypsin (ACT), are directed against hLE and hCG, respectively(Groutas, 1987). Imbalances in the levels of tissue proteases such ashLE and hCG, and their inhibitors, allow excess hLE and hCG to attackconnective tissue, and are implicated in the severe and permanent tissuedamage associated with pulmonary emphysema (Groutas, 1987), rheumatoidarthritis (Gallin et al., 1988), cystic fibrosis (Jackson et al., 1984)and several other inflammatory conditions. Major research efforts havebeen dedicated to develop potent inhibitors of hLE and hCG based on awide variety of low molecular weight organic compounds (Edwards andBernstein, 1994) such as 3,3-dialkylazetidin-2-ones, proposed as orallyactive β-lactam inhibitors of hLE (Finke et al., 1995).

CRP as a whole protein was reported to have no inhibitory effect on hLE(Vachino et al., 1988). In contrast, a specific region within theprimary sequence of CRP containing the core peptideVal₈₉-Thr-Val-Ala-Pro-Val-His-Ile₉₆ of SEQ ID NO:1 was shown to inhibitin vitro the enzymatic activities of hLE and hCG to a larger extent thanpeptides of similar chain lengths corresponding to the active sites oftheir natural inhibitors (PCT Publication No. WO 97/28182; Yavin et al.,1996). Novel biologically active CRP-derived peptides, i.e. peptidescapable of inhibiting in vitro the enzymatic activity of hLE and/or ofhCG, previously concealed within the inner hydrophobic disulfide loopwhich spans CRP36-97 in each subunit (see FIG. 1), have been found inaccordance with the present invention to significantly inhibit theenzymatic activities of hLE and hCG enzymes.

SUMMARY OF THE INVENTION

The present invention relates to a synthetic CRP-derived peptide capableof inhibiting in vitro the enzymatic activity of human leukocyteelastase (hLE) and/or of human cathepsin G (hCG), said peptide beingselected from:

(i) a core peptide corresponding to positions 62-71 of the sequence ofhuman C-reactive protein (CRP) of the formula:

Glu₆₂-Ile-Leu-Ile-Phe-Trp-Ser-Lys-Asp-Ile₇₁

or peptides resulting from modification thereof characterized by:

(ii) substitution of Glu₆₂ by Asp or by a non-amino acid negativelycharged residue derived from succinic, glutaric or adipic acids;

(iii) substitution of each of the residues Ile₆₃, Leu₆₄, Ile₆₅, Phe₆₆ orTrp₆₇ by a natural or non-natural hydrophobic amino acid residue;

(iv) deletion of 1 or 2 amino acid residues selected from Ile₆₃, Leu₆₄,Ile₆₅, Phe₆₆ and Trp₆₇;

(v) substitution of 1-3 amino acid residues selected from Ile₆₃, Leu₆₄,Ile₆₅, Phe₆₆ and Trp₆₇ by a single non-natural amino acid residuederived from 6-aminocaproic acid;

(vi) substitution of 2-4 amino acid residues selected from Ile₆₃, Leu₆₄,Ile₆₅, Phe₆₆ and Trp₆₇ by a single non-natural amino acid residuederived from 8-aminocaproic acid, 10-aminodecanoic acid or12-aminolauric acid;

(vii) substitution of 3-5 amino acid residues selected from Ile₆₃,Leu₆₄, Ile₆₅, Phe₆₆ and Trp₆₇ by a stretch of identical hydrophobicamino acid residues or by a single non-natural amino acid residuederived from 10-aminodecanoic acid or 12-aminolauric acid;

(viii) substitution of Ser₆₈ by a natural or non-natural amino acidresidue selected from Thr, Cys, Ala and homoserine;

(ix) substitution of Lys₆₉ by a natural or non-natural positivelycharged or hydrophobic amino acid residue;

(x) substitution of Asp₇₀ by a negatively charged or a polar amino acidresidue selected from Glu, Asn or Gln;

(xi) substitution of Ile₇₁ by a natural or non-natural hydrophobic aminoacid residue;

(xii) elongation of a peptide (i) to (xi) by 1-5 non-charged amino acidresidues at the N-terminus and/or at the C-terminus;

(xiii) substitution of any amino acid residue in a peptide (i) to (xii)by the corresponding N-alkyl derivative, D-amino acid residue or byanother isoster;

(xiv) an amide of the C-terminal of a peptide (i) to (xiii); and

(xv) an N-acyl derivative of a peptide (i) to (xiv).

The invention further relates to anti-inflammatory pharmaceuticalcompositions comprising a CRP-derived peptide of the invention and apharmaceutically acceptable carrier.

In another aspect, the invention relates to a method of treatment of aninflammatory disorder, e.g. rheumatoid arthritis, pulmonary emphysema,cystic fibrosis, bronchitis, asthma, acute respiratory distress syndromeand other chronic inflammatory tissue destructive conditions, whichcomprises administering to a patient in need thereof an effective amountof a CRP-derived peptide according to the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the primary sequence of human C-reactive protein (CRP)(SEQ ID NO:1). The two cystein residues (at positions 36 and 97) whichcomprise the single disulfide bond within each subunit are marked inbold letters.

FIG. 2 depicts the sequence alignment of the CRP-derived 15-merpeptides, herein designated peptides 1-10, within the disulfide loopregion of the CRP36-97 sequence of SEQ ID NO:1.

FIG. 3 depicts a schematic representation of hLE's (and possibly hCG's)subsites designated S₈-S₂′, interacting with a segment of peptide 6(amino acid residues Glu₆₂-Ile₇₁ of SEQ ID NO:1). The S₈-S₂′ notation isused to designate subsites on the surface of the enzyme with respect tothe amino acids in the peptide inhibitor (P₈-P₂′, respectively), and thetheoretical cleavage site on the peptide at the Lys₆₉-Asp₇₀ bond(denoted P₁-P₁′). The negative side chain of CRP's Glu₆₂ is shown toreside within the positive pocket of hLE and the positive side chain ofLys₆₉ is shown to reside within the major hydrophobic pocket of hLEwhich possesses considerable negative charge.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a series of synthetic peptides derivedfrom positions 62-71 of the sequence of CRP and to pharmaceuticalcompositions comprising them which are anti-inflammatory by inhibitingeither hLE or hCG activity, or both. These biologically active peptidesaccording to the invention can be used to inhibit hLE and/or hCG andthereby have utility in controlling tissue damage associated withchronic inflammation.

The CRP hydrophobic disulfide loop domain contains hydrophobic aminoacid stretches concealed within the intact protein and many of thepreviously described biologically active CRP-derived peptides originatefrom this region following CRP binding to neutrophils and subsequentproteolysis. A series of overlapping peptides derived from within theCRP's hydrophobic disulfide loop (CRP 36-97), herein designated peptides1-10, were synthesized according to the invention and are depicted inFIG. 2. Each successive peptide, 15 amino acids in length, overlapped in10-amino acid sequence with each one of its neighboring peptides. ThehLE and hCG inhibitory activities of these peptides were evaluated asshown in Examples 4 and 6 and Table 1 hereinafter.

It was found according to the invention that Peptide 6 corresponding topositions 62-76 of the CRP sequence possesses a unique inhibitoryactivity towards both hLE and hCG observed at submicromolarconcentrations of the peptide. Surprisingly, it was found that thisinhibitory activity is two orders of magnitude more potent than thepeptides which contain the sequences Val₈₉-Thr-Val-Ala-Pro-Val-His-Ile₉₆of SEQ ID NO:1 previously described in PCT Publication No. WO 97/28182and Yavin et al., 1996.

The unique inhibitory activity of peptide 6 (CRP62-76) was furtherstudied by synthesizing and examining the inhibitory effect of analogsand variants thereof in which one or more amino acid residues have beenadded, deleted or replaced by natural or non-natural amino acid residues(see Table 2, peptides 11-26).

A possible mode of binding of peptide 6 in which the negative charge ofGlu₆₂ residue interacts with hLE's (and possibly hCG's) positive pocket(Yavin et al., 1997) is depicted schematically in FIG. 3. In such asuggested binding configuration, the Lys₆₉ residue of CRP fits into hLEmajor negative-hydrophobic pocket with the hydrophobic amino acidsIle₆₃, Leu₆₄, Ile₆₅, Phe₆₆ and Trp₆₇ traversing the space between thetwo distant pockets and interacting with the enzyme's hydrophobicsurface.

Peptide 6 is remarkably resistant to proteolysis, it binds and inhibitsboth hLE and hCG but is not susceptible to proteolytic inactivation bythem. The stability of the peptide bonds in peptide 6 were evaluated byincubating the peptide with hLE and hCG and monitoring proteolysis byreverse-phase HPLC chromatography analysis. Surprisingly, no cleavageproducts were observed using the degradation conditions as described byYavin et al., 1996, even after 12 hours of the peptide incubation at 37°C.

In order to determine the significance of the amino acid residues atspecific positions with respect to the inhibitory activity of peptide 6,short peptides obtained by deletion of amino acid residues from the N-and/or C-terminus of peptide 6 (see Table 2, peptides 11-16) werecarefully studied for their inhibitory effects.

The residue Glu₆₂ is the preferred amino acid residue at this position;a dramatic loss in both hLE and hCG inhibitory activities is observedwhen the N-terminus Glu₆₂ is removed (peptide 11) indicating the crucialrole this negatively charged residue plays. It can however be replacedby another negatively charged amino acid such as Asp (peptide 18) or bya non-amino acid negatively charged residue such as a residue derivedfrom succinic, glutaric or adipic acids. The negative charge of thesubstituting residue is important for the inhibitory activity of thepeptide, as well as the fitting of the substituting residue into theenzyme's positive pocket.

The hydrophobic amino acid residues Ile₆₃ and Leu₆₄ are critical inestablishing hydrophobic interactions needed for efficient binding.However, the residues Ile₆₃, Leu₆₄, Ile₆₅, Phe₆₆ and Trp₆₇ of peptide 6may be interchanged or replaced by natural or non-natural hydrophobicamino acid residues selected from Ala, Val, Leu, Ile, Phe, Trp, Tyr, Nvaand Nle. One to five of the amino acid residues of this stretch may besubstituted by a stretch of identical amino acid residues as indicatedbefore, e.g. a stretch of 4 or 5 Ala residues, or by a non-natural aminoacid residue spacer derived from 6-aminocaproic acid, 8-aminocaproicacid, 10-aminodecanoic acid or 12-aminolauric acid, with the provisothat the overall length of this stretch is not significantly changed.For example, 1-3 amino acid residues may be substituted by a singleamino acid residue derived from 6-aminocaproic acid, 2-4 amino acidresidues may be substituted by a single amino acid residue derived from8-aminocaproic acid or 10-aminodecanoic acid, and 3-5 amino acidresidues may be substituted by a stretch of 3-5 Ala residues or by asingle amino acid residue derived from 10-aminodecanoic acid or12-aminolauric acid. Minor deletions (up to two amino acid residues)within this hydrophobic stretch are also possible, still allowing thebinding of both charges into the respective charged pockets on thesurface of both hLE and hCG enzymes (FIG. 3).

The residue Ser₆₈ allows flexibility in contrast to proline which wasobserved at the S₂ position of many inhibitors, including the previouslydescribed CRP derived core peptide Val₈₉-Thr-Val-Ala-Pro-Val-His-Ile₉₆of SEQ ID NO:1 (PCT Publication No. WO 97/28182; Yavin et al., 1996).This residue may be substituted by a similar compact amino acid such asThr, Cys or Ala, or by a non-natural isoster such as homoserine.

Peptide 6 is less sensitive to removal of amino acids at the C-terminus.Removing the first three amino acids at end (peptide 13) results indiminished inhibitory activity towards both enzymes, yet less than theloss of activity observed with the removal of the single N-terminusamino acid residue Glu₆₂. Furthermore, peptide 6 may tolerate theremoval of five C-terminus amino acid residues (peptide 14) and stillretain significant inhibitory activity, indicating that the interactionof this part of the peptide with both enzymes is less specific incomparison to the charged amino acids and hydrophobic section(Ile₆₃-Trp₆₇).

The residue Lys₆₉ is the preferred residue at this position, yet it maybe replaced by another positively charged amino acid residue such asArg, His, homolysine, ornithine (Orn) or diaminobutyric acid (DAB) or byan hydrophobic amino acid such as Ala, Val, Leu, Ile, Phe, Nva or Nle.Bulky amino acids such as Phe, Tyr and His are preferred amino acidresidues at this position (P₁) in hCG inhibitors (Yavin et al., 1997).Modification of Lys₆₉ as described above may lead to peptide bondcleavage by both enzymes in which case it is advisable to use D-aminoacids at this position.

The Asp₇₀ and Ile₇₁ residues are important in establishing a strong fitwith both hLE's and hCG's surfaces. A negatively charged residue atposition Asp₇₀ is essential for the peptide inhibitory activity towardshLE and hCG.

The residue Asp₇₀ is the preferred residue at this position, yet it maybe replaced by a negatively charged or a polar amino acid residue suchas Glu, Asn or Gln. Modification of this residue may lead to peptidebond cleavage by both enzymes in which case it is advisable to useD-amino acids or preferably N-alkyl derivatives of the substitutingresidue.

The residue Ile₇l is the preferred residue at this position, yet it maybe replaced by a large hydrophobic amino acid residue such as Leu, Ile,Phe and Tyr, or by an isoster such as Nva, Nle, homoleucine,homoisoleucine and aminobutyric acid (ABU).

Elongation of peptide 6 by one to five non-charged amino acid residuesat the N- and/or C-terminus does not impair its inhibitory activity, buta more extended elongation, for example, by addition of amino acidresidues according to the sequence of CRP at both the N- and C-terminussuch as in peptide 17, leads to a reduction of inhibitory activitytowards both hLE and hCG. This decrease in activity may arise from anunfavorable conformation or folding of the 29-amino acid peptide.

The residues Gly₇₂, Tyr₇₃, Ser₇₄, Phe₇₅ and Thr₇₆ may be replaced bynon-charged amino acid residues and are less critical in establishingpotent inhibition.

As a general rule, additional charged amino acids besides in thespecific positions of Glu₆₂ and Lys₆₉ should be avoided such as not tocause a gross misalignment of the peptide inhibitor on the enzyme'ssurface (see FIG. 3).

Amides (CO—NH₂) of the carboxy terminal of the peptides of the inventionare also encompassed by the invention and show an inhibitory activitytowards hLE and hCG as well as N-acyl derivatives of the N-terminal suchas those corresponding to the formula R—X—CO— wherein R is a substitutedor unsubstituted hydrocarbyl, preferably alkyl or aryl, and X is acovalent bond, O, NH or NHCO. Examples of acyl radicals are octanoyl,monomethoxysuccinyl, acetylaminocaproyl, adamantyl-NH—CO—, and morepreferably, carbobenzoxy (benzyl-O—CO—), naphthyl-NH—CO—, and Fmoc(fluorenylmethyl-O—CO—).

The peptides according to the invention have a hLE K_(i) lower of about8.0 μM, preferably 0.1-3.0 μM. Thus, preferred CRP-derived peptidesaccording to the invention are the peptides 6, 13, 18, 20-23 and 25-26.

The peptides of the invention are prepared by standard methods for thesynthesis of peptides, for example as set forth in the Exampleshereinbelow.

In another aspect, the present invention relates to pharmaceuticalcompositions comprising a peptide of the invention and apharmaceutically acceptable carrier. The compositions are prepared bywell-accepted methods for preparation of peptide-containingpharmaceutical compositions for administration in a suitable form, e.g.orally, subcutaneously, intranasal, and parenterally includingintravenous, intramuscular and intraperitoneally, according to theinflammatory condition to be treated.

In a further aspect, the invention relates to a method of treatment of achronic inflammatory condition which comprises administering to apatient in need thereof an effective amount of a peptide according tothe invention. Examples of such chronic inflammatory conditions arerheumatoid arthritis, pulmonary emphysema, cystic fibrosis, bronchitis,asthma and acute respiratory distress syndrome. The anti-inflammnatorypeptide is administered and dosed in accordance with good medicalpractice, taking into consideration the clinical condition of thepatient, the site and method of administration, schedule ofadministration and other factors known to medical practitioners.

The invention will now be illustrated by the following non-limitingexamples.

EXAMPLES Materials and Methods

(i) General Solid Phase Peptide Synthesis: Peptides were prepared byconventional solid phase peptide synthesis, with ABIMED AMS422 automatedsolid phase multiple peptide synthesizer (Langenfeld, Germany). TheFmoc-strategy (9-fluorenyl-methoxycarbonyl) was used through peptidechain assembly, following the company's commercial protocols. In eachreaction vessel, 12.5 μmol of Wang resin was used which contained thefirst, covalently bound, corresponding N-Fmoc C-terminal amino acid(typical polymer loadings of 0.3-0.7 mmols/g resin were employed). Fmocdeprotection was achieved using duplicate treatments with 20% piperidinein dimethylformamide (DMF), typically for 10-15 min at room temperature,depending on the length of peptide and Fmoc- protected amino acid type,as given by the company's protocols.

Side chain-protecting groups were tert.-butyloxycarbonyl (t.-Boc) forLys, diaminobutyric-acid (DAB), and Trp; trityl (Trt) for Asn, Cys, Gln,His, and (D)-His; tert.-butyl-ester (O-t-But) for Asp and Glu;tert.-butyl-ether (t-But) for Ser, Thr, and Tyr.

Coupling was achieved, as a rule, using two successive reactions with 50μmol (4 eqv.) of corresponding N-Fmoc protected amino acid, 50 μmol (4eqv.) of PyBOP reagent(benzotriazole-1-oxytris-pyrrolldino-phosphonium-hexafluoro-phosphate),and 100 μmol (μeqv.) of 4-methyl-morpholine (NMM), all dissolved in DMF,typically for 20-45 min at room temperature, depending on the length ofpeptide and amino acid derivative type, as given by the company'sprotocols.

Cleavage of the peptide from the polymer was achieved by reacting theresin with trifluoroacetic acid/H₂O/triethylsilane (TFA/H₂O/TES; 90/5/5;v/v) for 1.5 to 2 hours at room temperature In the cleavage of peptideswhich contained Arg and Trp (peptide 5), the cleavage mixture wascomposed of TFA/H₂O/ethanediol/thioanisol/cresol (80/5/5/5/5-v/v). Therespective solutions containing the crude.unprotected peptides were thencooled down to 4° C., precipitated with ice-cold di-tert.-butylether(DTBE) and centrifuged for 15 min, 3000 RPM at 4° C. The pellet waswashed and centrifuged 3 times with DTBE, dissolved in 30% acetonitrilein H₂O, and lyophilized.

All protected amino acids, coupling reagents, and polymers were obtainedfrom Nova Biochemicals (Laufelfingen, Switzerland). Synthesis-gradesolvents were obtained from Labscan (Dublin, Ireland).

(ii) Reversed-chase high performance liquid chromatography (RP-HPLC):Synthetic peptides were purified by using a prepacked LiChroCart RP-18column (250×10 mm, 7 μm bead size), employing a binary gradient formedfrom 0.1% TFA in H₂O (solution A) and 0.1% TFA with 25% H2O inacetonitrile (solution B), eluted at t=0 min B=5% t=5 min B=5% t=60 minB=70% (flow-rate 5 mL/min). Analytical RP-HPLC was performed using aprepacked Lichrospher-100 RP-18 column (4×250 mm, 5 μm bead size) usingthe same buffer system (flow-rate 0.8 mL/min). All peptide separationswere performed using a Spectra-Physics SP8800 liquid chromatographysystem equipped with an Applied Biosystems 757 variable wave-lengthabsorbance detector. The column effluents were monitored by UVabsorbance at 220 nm, and chromatograms were recorded on a Chrome-Jetintegrator. Following HPLC purification, the lyophilized peptides(generally >90% pure for crude samples after synthesis as describedbelow) were purified to >97%. All solvents and HPLC columns wereobtained from Merck (Darmstadt, Germany).

(iii) Amino acid composition analysis: Purified peptide solutions wereroto-evaporated (˜40 μg of peptide in 40 μL solution with 5 μg ofnorleucine as an unnatural amino acid internal standard), hydrolyzed in6 N HCl at 110° C. for 22 hours under vacuum and analyzed with a Dionexamino acid analyzer. This quantification was used as a basis fordetermination of the total yield of peptide. Several of the peptidessynthesized were analyzed by Electrospray mass-spectrometry whichconfirmed their expected molecular weights.

(iv) Isolation of hLE and hCG: The isolation of neutrophilic enzymes wasbased on the two-step aprotinin-sepharose affinity chromatography andcarboxymethyl-cellulose (CMC) ion exchange chromatography (Heck et al.,1985). Neutrophils (1.4 billion) were isolated from whole blood obtainedfrom a single healthy laboratory donor by dextran sedimentation andFicoll/hypaque gradient centrifugations as described elsewhere (Metcalfet al., 1986). The enzymatic activity was assayed with MeOSuc-AAPV-NAfor hLE determination and Suc-AAPF-NA for hCG determination (both in 100mM Hepes buffer, pH 7.4, containing 0.05% of the anionic detergentBrij-35). The activities of the individual enzymatic fractions were 100%free from cross-contamination. The step-wise elution profile on the CMCcolumn with a long 0.45 M NaCl elution step (20 column volumes) affordedthe effective separation between the two enzymes. The fractionscontaining hLE and hCG were dialyzed each against 0.1% pyridiniumacetate, pH 5.3, divided into 20 aliquots, lyophilized, and stored at−20 ° C. until use. By the initial rates of reactions and the knownvalues of K_(cat) (hLE=54 μM, hCG=2900 μM) and K_(m) (hLE=13.3 sec.⁻¹,hCG=3.1 sec.⁻¹), the amount of enzyme was estimated to be approximately15 μg/aliquot for hLE and 12 μg/aliquot for hCG, such values beingconfirmed by active site titration with α₁-protease inhibitor andα-antichymotrypsin.

(v) Inhibition experiments with hLE: Peptides were dissolved in 100 mMHepes buffer pH 7.4 containing 0.1% Brij-35 with 10%. DMSO to yield 300μM solutions, which were used to make further dilutions with the samebuffer, and 80 μL aliquots were added in duplicates to 96-well plates.The substrate, 600 μM MeOSuc-AAPV-NA in the same buffer with 5% DMSO,was added to each well in addition to the blank wells, and the plate wasplaced in the plate reader equilibrated to 37 C (Dynateck MR-6000).Lyophilized aliquots of hLE were dissolved in 1600 μL of 100 mM Hepesbuffer without DMSO, and 80 μL of the enzyme solution was added to thepeptides and substrate to initiate the reaction. The kinetics programread the plate at 405 nm every 2 min for 20 min (with a 3 sec shakingperiod between readings), and plotted the results as well as the averageof each duplicate. The final volume was 240 μL containing: 5% DMSO,1-100 μM of peptide, 200 μM substrate, and 0.75 μg (˜25 picomol) enzyme.

vi) Inhibition experiments with hCG: Similar conditions to hLEinhibition experiments were used except the substrate: 80 μL of 1.80 mMSuc-AAPF-NA. The enzyme was dissolved in 800 μL buffer, and the reactionwas monitored every 6 min for 1 hour. The final volume was 240 μLcontaining: 5% DMSO, 1-100 μM peptide, 600 μM substrate, and 1.2 μgenzyme (about 40 picomol).

(vii) Degradation profiles of peptides by RP-HPLC: Several activepeptide inhibitors were dissolved in calcium- and magnesium-freephosphate-buffered saline (PBS), 125 μg /250 μL, mixed with 0.25aliquots of hLE or hCG in 250 μL PBS and incubated at 37° C.Periodically (up to 12 hrs), 100 μL samples were removed from thereaction vessel. The samples were diluted with 150 μL of 0.1% TFA,frozen with liquid nitrogen, and stored at −20° C. prior to HPLCanalysis.

(viii) Calculations: For hLE, V is determined by fitting a linearequation to the first 6 time-points (10 min) of the kinetics data usingthe least squares method. Without exception, all R² factors were >0.998.Several inhibitor concentration in duplicates and two control wells wereused to fit a linear equation to graphs of V_(o)/V_(i)−1 vs. [I] foreach inhibitor using the least squares method (8 data points for eachinhibitor). From calculating the error in the slope of the equation, therelative error for K_(i) was deduced:

K _(i)={slope*(1+[S]/K _(m))}⁻¹because K _(i) =[I]*{(1+[S]/K _(m))*(Vo/V_(i−1))}⁻¹.

For hCG, V is determined by fitting a quadratic equation to the totalkinetic data (60 min), using the least squares method and calculating Vat t=0. Without exception, all R² factors were >0.996. Two inhibitorconcentrations in duplicate and two control wells were used for eachinhibitor, and in a similar fashion to hLE, K_(i) was deduced (6 datapoints for each inhibitor).

EXAMPLE 1

Synthesis of Peptides 1-26

In the synthesis of peptide 6.Glu₆₂-Ile-Leu-Ile-Phe-Trp-Ser-Lys-Asp-Ile-Gly-Tyr-Ser-Phe-Thr₇₆, thestandard Fmoc protocol was used as follows:

Peptide Elongation Cycle:

Step 1. DMF wash x6 Step 2. Deprotection: 20% piperidine in DMF x2 Step3. DMF wash x6 Step 4. Derivative coupling. x2

At the end of Synthesis:

Step 1. DMF wash x6 Step 2. Deprotection: 20% piperidine in DMF x2 Step3. DMF wash x6 Step 4. CH₂Cl₂ wash x6

Deprotection, coupling and wash times and volumes, were calculated bythe ABIMED computer program. The resulting lyophilized crude peptide waspurified by preparative HPLC to yield approx. 15 mg of lyophilizedpeptide (white powder), above 98% pure, as determined by its analyticalRP-HPLC. Amino acid analysis confirmed the expected 'sequence, purity,and yield of purified peptide (see Table 3).

Additional peptides 5, 11, 13, 14, 17, 18, 20-23, 25-26 of the inventionand comparison peptides 1-5, 7, 10, 12, 15, 16, 19 and 24 weresynthesized in a similar manner using the appropriate amino acidresidues.

The sequence of the peptides 1-26 and their inhibition constants (K_(i))of human hLE and human hCG are shown in Tables 1 and 2. The amino acidanalysis of the same peptides are shown in Table 3.

EXAMPLE 2

Synthesis of C-terminus Amides of the Peptides

C-terminus amidated peptides (peptide-NH₂) are prepared according topreviously described procedures (PCT Publication No. WO 97/28182; Yavinet al., 1996): The standard resin is replaced with rink amide solidsupport [4-2′(4′-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxy-resin] whichdoes not contain the first amino acid. Peptide synthesis is followed inan identical fashion as described in Example 1 above, and upon cleavagefrom the polymer, the carboxy terminus amidated form of the peptide isobtained.

TABLE 1 Amino acid sequence of CRP-derived peptides 1-10 and theirinhibition constants (Ki) of hLE and hCG. Peptide Sequence hLE Ki [μM]hCG Ki [μM]  1 CRP₃₇₋₅₁ Leu-His-Phe-Tyr-Thr-Glu-Leu-Ser-Ser-Thr-Arg-w.i. n.s.i. Gly-Tyr-Ser-Ile  2 CRP₄₂₋₅₆Glu-Leu-Ser-Ser-Thr-Arg-Gly-Tyr-Ser- n.s.i. n.s.i.Ile-Phe-Ser-Tyr-Ala-Thr  3 CRP₄₇₋₆₁Arg-Gly-Tyr-Ser-Ile-Phe-Ser-Tyr-Ala-Thr- n.s.i. n.s.i.Lys-Arg-Gln-Asp-Asn  4 CRP₅₂₋₆₆ Phe-Ser-Tyr-Ala-Thr-Lys-Arg-Gln-Asp-n.s.i. w.i. Asn-Glu-Ile-Leu-Ile-Phe  5 CRP₅₇₋₇₁Lys-Arg-Gln-Asp-Asn-Glu-Ile-Leu-Ile-Phe-Trp- 85 ± 10 180 ± 25 Ser-Lys-Asp-Ile  6 CRP₆₂₋₇₆ Glu-Ile-Leu-Ile-Phe-Trp-Ser-Lys-Asp-Ile-Gly-0.18 ± 0.03 0.25 ± 0.05 Tyr-Ser-Phe-Thr  7 CRP₆₇₋₈₁Trp-Ser-Lys-Asp-Ile-Gly-Tyr-Ser-Phe-Thr-Val- n.s.i. w.i. Gly-Gly-Ser-Glu 8 CRP₇₂₋₈₆ Gly-Tyr-Ser-Phe-Thr-Val-Gly-Gly-Ser-Glu-Ile- n.s.i. w.i.Leu-Phe-Glu-Val  9 CRP₇₇₋₉₁ Val-Gly-Gly-Ser-Glu-Ile-Leu-Phe-Glu-Val-Pro-n.s.i. w.i. Glu-Val-Thr-Val 10 CRP₈₂₋₉₆Ile-Leu-Phe-Glu-Val-Pro-Glu-Val-Thr-Val-Ala- 55 ± 5  150 ± 15 Pro-Val-His-Ile

Each one of peptides 1-10 corresponds to a 15-mer within the stretch ofCRP36-96. Subscript numbers in column 1 denote the residue positions ofeach peptide within the primary sequence of CRP (SEQ ID NO:1). w.i.;weak inhibition (>200 μM). n.s.i.; no significant inhibition.

TABLE 2 Amino acid sequence of CRP-derived peptides and analogs 6, 11-30and their inhibition constants (Ki) of hLE and hCG. Peptide Sequence hLEKi [μM] hCG Ki [μM]  6 CRP₆₂₋₇₆ Glu-Ile-Leu-Ile-Phe-Trp-Ser-Lys-Asp-0.18 ± 0.03 0.25 ± 0.05 Ile-Gly-Tyr-Ser-Phe-Thr 11 CRP₆₃₋₇₆Ile-Leu-Ile-Phe-Trp-Ser-Lys-Asp-Ile- 4.5 ± 0.5 15 ± 4 Gly-Tyr-Ser-Phe-Thr 12 CRP₆₅₋₇₆ Ile-Phe-Trp-Ser-Lys-Asp-Ile-Gly-Tyr-n.s.i. n.s.i. Ser-Phe-Thr 13 CRP₆₂₋₇₃Glu-Ile-Leu-Ile-Phe-Trp-Ser-Lys-Asp- 1.1 ± 0.2 4.0 ± 0.6 Ile-Gly-Tyr 14CRP₆₂₋₇₁ Glu-Ile-Leu-Ile-Phe-Trp-Ser-Lys-Asp-Ile 6.7 ± 1.0 6.0 ± 1.0 15CRP₆₂₋₆₉ Glu-Ile-Leu-Ile-Phe-Trp-Ser-Lys n.s.i. n.s.i. 16 CRP₆₈₋₇₄Ser-Lys-Asp-Ile-Gly-Tyr-Ser n.s.i. n.s.i. 17 CRP₅₅₋₈₃Ala-Thr-Lys-Arg-Gln-Asp-Asn-Glu-Ile- 7.3 ± 1.0 5.5 ± 1.0Leu-Ile-Phe-Trp-Ser-Lys-Asp-Ile-Gly-Tyr-Ser-Phe-Thr-Val-Gly-Gly-Ser-Glu-Ile-Leu 18Asp-Ile-Leu-Ile-Phe-Trp-Ser-Lys-Asp- 2.2 ± 0.4 2.6 ± 0.5Ile-Gly-Tyr-Ser-Phe-Thr 19 Glu-Ile-Leu-Ile-Phe-Trp-Pro-Lys-Asp- n.s.i.30 ± 5  Ile-Gly-Tyr-Ser-Phe-Thr 20 Glu-Ile-Leu-Ile-Phe-Trp-Ser-Orn-Asp-0.25 ± 0.10 0.25 ± 0.05 Ile-Gly-Tyr-Ser-Phe-Thr 21Glu-Ile-Leu-Ile-Phe-Trp-Ser-DAB-Asp- 0.20 ± 0.05 0.85 ± 0.35Ile-Gly-Tyr-Ser-Phe-Thr 22 Glu-Ile-Leu-Ile-Phe-Trp-Ser-Lys-Ala- 3.0 ±0.6 5.5 ± 1.5 Ile-Gly-Tyr-Ser-Phe-Thr 23Glu-Ile-Leu-Ile-Phe-Trp-Ser-Lys-Glu- 0.55 ± 0.15 0.85 ± 0.25Ile-Gly-Tyr-Ser-Phe-Thr 24 Thr-Phe-Ser-Tyr-Gly-Ile-Asp-Lys-Ser-Trp- 10 ±2  — Phe-Ile-Leu-Ile-Glu (reverse sequence) 25Glu-Ile-Leu-Ile-Phe-Trp-Ser-Ala-Asp-Ile 3.0 ± 0.5 — 26Glu-Ile-Leu-Ile-Phe-Trp-Ser-Val-Asp-Ile 2.0 ± 0.5 —

Peptides 11-26 are analogs and variants of peptide 6 (CRP₆₂₋₇₆)Subscript numbers in column 1 denote the residue positions of thepeptide within the primary sequence of CRP (SEQ ID NO:1). Peptides 18-26correspond to SEQ ID NOs:4-12, respectively. Substituted amino acids areunderlined. w.i.; weak inhibition (>200 μM). n.s.i.; no significantinhibition, undetermined. Error margins were calculated for eachinhibitor as described in Material and Methods section viii.

TABLE 3 The amino acid analysis ratios and HPLC retention times (R.T.)of peptides 1-26. The theoretical values are without exception thoseobtained by rounding off each value to the closest integer. Pep. Asp/nGlu/n Ser His Gly Thr Ala Arg Tyr Val Ile Phe Leu Lys* Pro R.T. 1 — 0.952.80 1.00 1.05 1.85 — 1.00 1.90 — 0.95 0.95 1.90 — — 25.8 2 — 0.95 4.00— 1.05 1.95 1.00 1.00 2.00 — 1.00 1.00 1.00 — — 25.8 3 1.95 1.00 1.95 —1.00 0.90 1.00 2.00 1.95 — 1.00 1.00 — 1.05  — 22.7 4 2.00 2.00 1.00 — —0.95 1.00 1.00 0.95 — 2.00 2.00 1.00 1.10  — 28.7 5 2.95 1.90 1.05 — — —— 1.00 — — 3.20 1.00 1.00 2.15  — 29.8 6 1.00 1.05 2.00 — 1.00 0.95 — —0.95 — 3.10 2.00 1.00 0.95  — 31.9 7 1.05 1.00 3.00 — 3.25 1.00 — — 1.001.00 1.00 1.00 — 1.10  — 23.4 8 — 2.00 2.00 — 3.10 0.95 — — 1.00 2.001.00 2.00 1.00 — — 30.8 9 — 3.00 1.00 — 2.10 0.95 — — — 4.00 1.00 1.001.00 — 1.00 29.2 10 — 2.00 — 1.10 — 0.95 1.05 — — 3.95 2.00 1.00 1.00 —1.95 30.9 11 1.05 — 2.05 — 1.05 0.95 — — 0.95 — 3.05 2.00 1.00 0.95  —32.6 12 1.00 — 2.00 — 1.05 1.00 — — 0.95 — 2.15 2.05 — 1.00  — 28.5 131.00 1.00 1.05 — 1.00 — — — 0.95 — 3.20 1.00 1.00 1.00  — 32.0 14 1.150.85 1.10 — — — — — — — 3.30 1.00 1.00 1.00  — 31.1 15 — 1.00 1.00 — — —— — — — 2.15 1.00 1.00 1.00  — 30.2 16 1.00 — 2.00 — 1.10 — — — 1.00 —1.00 — — 1.15  — 13.5 17 2.95 2.95 2.80 — 3.25 1.85 1.00 1.00 0.90 1.004.10 2.00 2.00 2.20  — 32.4 18 2.05 — 2.00 — 1.05 0.95 — — 1.00 — 3.002.10 1.00 1.05  — 33.1 19 1.05 0.95 0.90 — 1.10 1.00 — — 1.00 — 3.002.21 1.00 1.15  1.00 33.6 20 1.10 1.00 1.95 — 1.05 1.00 — — 1.00 — 3.002.10 1.05 1.05* — 32.6 21 1.05 1.00 1.90 — 1.05 0.95 — — 0.95 — 3.001.95 1.00 1.00* — 32.7 22 — 1.00 1.90 — 1.10 1.00 1.05 — 1.05 — 3.002.05 1.00 1.05  — 33.4 23 — 2.05 1.80 — 1.10 1.00 — — 1.05 — 3.00 2.000.95 1.05  — 32.8 24 1.05 1.00 2.00 — 0.95 0.95 — — 1.00 — 3.05 2.050.95 1.00  — 32.2 25 1.05 0.85 1.00 — — — 1.00 — — — 3.15 1.05 1.00 — —32.7 26 1.00 0.95 1.05 — — — — — — 1.05 3.05 1.00 0.95 — — 32.9 *Inpeptides 20 and 21, (L)-ornithine (Orn) and 1,4-(L)-diaminobutyric-acid(DAB) respectively.

EXAMPLE 3

Synthesis of N-acyl Peptides

N-terminus acylated peptides (R-CO-peptide) are prepared according topreviously described procedures (PCT Publication No. WO 97/28182; Yavinet al., 1996): Several organic compounds which contain a free carboxylicacid moiety are used for coupling to the exposed N-terminus of thepeptides as the final step of solid phase peptide synthesis prior topeptide-polymer cleavage and deprotection. Examples of such organiccompounds are: mono-methyl-succinic-acid, CH₃OCO(CH₂)₂COOH,CH₃(CH₂)₆COOH and N-acetyl-amino-caproic acid. PyBOP and NMM coupling isused as described in the Materials and Methods section (i) followed byextensive flushing with N-methyl-pyrrolidone (NMP) and CH₂Cl₂.

EXAMPLE 4

Synthesis of N-terminus Fmoc Peptides

The synthesis of Fmoc peptides is carried out as in Example 1 aboveexcept that the final step of Fmoc deprotection is omitted. The Fmocmoiety is stable under peptide-polymer cleavage and side-chaindeprotection conditions, thus yielding N-terminus Fmoc-peptides as theend products of the synthesis.

EXAMPLE 5

In Vitro Inhibition of hLE by the Peptides of the Invention

The hLE inhibitory capability of CRP-derived peptides was evaluated byinhibiting the enzymatic cleavage of MeOSuc-AAPV-NA as described inMaterials and Methods (section v). The results are shown in Tables 1 and2.

As shown in Table 1, the CRP-derived peptide 6 is extremely potent ininhibiting hLE. Total inhibition was observed at concentrations above 50μM and only by diluting the peptide to the 1-10 μM range, thedetermination of a K_(i) value for peptide 6 was made possible.

In contrast, from the other 9 peptides of the series 1-10, derived fromoverlapping sequences within CRP36-97, only peptides 5 and 10 displayedhLE inhibitory activity in the 50-100 μM range, still two orders ofmagnitude less potent than peptide 6, while peptides 14 and 7-9 wereinactive.

Peptides 11-16 shown in Table 2 represent short peptides in which 1-7amino acid residues from the N- and/or C-terminus of peptide 6 weredeleted. The most dramatic effect due to removal of a single amino acidresidue was observed when the Glu₆₂ residue was removed (peptide 11)resulting in around 20 fold decrease in hLE inhibition. Further removalof the two subsequent amino acid residues Ile₆₃ and Leu₆₄ totallyabolished the inhibitory activity (peptide 12).

Removal of amino acid residues at the C-terminus of peptide 6 was lesseffective in impairing the peptide inhibitory activity. Removal of thefirst three amino acids at the C-terminus of peptide 6 (peptide 13)resulted in diminished hLE inhibitory activity, albeit less than theloss of activity observed with the removal of the N-terminus residueGlu₆₂. Furthermore, peptide 14, that lacks the five C-terminus aminoacid residues of peptide 6, still retains significant inhibitoryactivity.

Peptide 15, that further lacks the Asp₇₀ and Ile₇l residues, showed atotal loss of inhibitory activity. As expected, peptide 16, missing boththe C-terminus and the critical N-terminus amino acid residues, was alsototally inactive.

Peptide 17, which is a 29-mer peptide (CRP₅₅₋₈₃) containing the aminoacid sequence of peptide 6 and additional amino acid residues accordingto the CRP sequence at its N- and C-terminus, has a reduced inhibitoryactivity which may result from an unfavorable conformation or folding ofthis longer peptide. In this respect, a comparison between peptide 5 andpeptide 14 confirms that the additional sequence of amino acidsLys₅₇-Arg-Gln-Asp-Asn₆₁ added to the N-terminus of peptide 14drastically lowered the inhibitory capability by more than one order ofmagnitude.

In order to evaluate the significance of a specific amino acid residueat a particular position with respect to the inhibitory activity ofpeptide 6, the substitution analogs peptides 18-26 were examined.Peptide 18, in which Glu₆₂ was substituted by the smaller negativelycharged amino acid Asp, shows a 10-fold lower inhibitory activitycomparing to peptide 6, supporting the notion that the stericarrangement of the negatively charged residue at this position is ofmajor importance.

Peptide 19, in which Ser₆₈ was replaced by Pro, showed a dramatic twoorders of magnitude loss of inhibitory activity (see Table 2). This lossmay be due to the bend introduced by the Pro amino acid residue in themiddle of the peptide preventing a good fit between the adjacent Lys₆₉and the S₁ pocket (FIG. 3).

Peptides 20 and 21 in which Lys₆₉ was substituted, respectively, byeither ornithine or DAB, retained the same level of inhibitory activityas peptide 6. Both ornithine and DAB are small positively chargedresidues fitting in the replaced Lys₆₉ position without interfering withthe peptide structure and interaction with the inhibited enzyme.

Substitution of Asp₇₀ by another negatively charged amino acid residueGlu (peptide 23) resulted in a small decrease in inhibitory activity,while substitution at the same position by Ala (peptide 22), anon-charged amino acid residue, resulted in a large decrease ininhibition capacity, stressing the importance of a negative charge atthis position.

The reverse sequence of peptide 6 (peptide 24), is 30 fold less activetowards hLE inhibition, stressing the importance of the precise bindingconformation as depicted in FIG. 3.

In peptides 25 and 26, the Lys₆₉ residue was substituted by smallhydrophobic amino acids (Ala and Val, respectively) to yield inhibitorswhich are slightly more potent than peptide 14.

EXAMPLE 6

In Vitro Inhibition of hCG by the Peptides of the Invention

The hCG inhibitory capability of CRP-derived peptides was evaluated byinhibiting the enzymatic cleavage of Suc-AAPF-NA as described inMaterials and Methods (section vi). The results are shown in Tables 1and 2.

The CRP-derived peptide 6, was shown to be extremely potent ininhibiting hCG. At concentrations above 50 μM, total inhibition wasobserved. In contrast, from the other 9 peptides of the series 1-10,derived from overlapping sequences within CRP36-97 (FIG. 2), onlypeptides 5 and 10 displayed hCG inhibitory activity in the 150-200 μMrange, 600-800 folds less potent than peptide 6, while peptides 1-4 and7-9 were inactive.

According to the invention it is shown that modifications of the corepeptide 6 examined in peptides 11-23 lead to similar effects on the hCGas well as on the hLE inhibitory activity. The same conclusions drawnfor the significance of each amino acid residue at its particularposition with respect to the inhibitory activity towards hLE generallyapply for the inhibitory activity towards hCG.

EXAMPLE 7

Evaluation of Peptide 6 Resistance to Proteolysis

Peptide 6 exhibits a remarkably resistance to proteolysis; it binds andinhibits both hLE and hCG but is not susceptible to proteolyticinactivation by them. The stability of the peptide bonds in peptide 6were evaluated by incubating the peptide with hLE and hCG and monitoringdegradation profiles of the peptide by reverse-phase HPLC. Briefly,peptide 6 was dissolved in calcium- and magnesium-freephosphate-buffered saline (PBS), 50 μg/250 μL, mixed with 3 μg of hLE orhCG in 250 μL PBS and incubated at 37° C. Periodically, after 1,3,8, and12 hrs incubation, 100 μL samples were removed from the reaction vessel.The samples were diluted with 150 μL of 0.1% TFA, frozen with liquidnitrogen, and stored at −20° C. prior to HPLC analysis. Surprisingly, nocleavage products were observed even after 12 hours of the peptide 6incubation at 37° C.

REFERENCES

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2. Bode, B., Meyer, E. & Powers, C. J. (1989) Human leukocyte elastaseand porcine pancreatic elastase: X-ray crystal structures, mechanism,substrate specifity and mechanism based inhibitors. Biochemistry, 28(5),1951-1963.

3. Edwards, P. D., Bernstein, P. R. (1994) Synthetic inhibitors ofElastase. Med. Res. Rev. 14, 127-194 and references cited therein.

4. Finke, P. E. et al., (1995) Orally active β-lactam inhibitors ofhuman leukocyte elastase. 3. Stereospecific synthesis andstructure--Activity relationships for 3,3-dialkylazetidin-2-ones. J.Med. Chem. 38, 2449-2462.

5. Gallin, J. I., Goldstein, I. M. & Snyderman, R. (1988) Inflammation.Chapter 41, Pathogenesis of rheumatoid arthritis: A disorder associatedwith dysfunctional immunoregulation. 751-774, ISBN 008167344-7.

6. Groutas, W. C. (1987) Inhibitors of leukocyte elastase and leukocytecathepsin G. Agents for the treatment of emphysema and related ailments.Medicinal Researh Reviews, 7(2), 227-241.

7. Heck, H. L., Darby, W. L., Bhown, A., Miller, E. J., Bennet, J. C.(1985) Isolation, characterization, and amino terminal amino acidsequence of human leukocyte elastase from normal donors. AnalyticalBiochemistry, 149, 153—162.

8. Jackson, A. H., Hill, S. L., Afford, S. C., Stockley, R. A. (1984)Sputum soluble phase proteins and elastase activity in patients withcystic fibrosis. J. Respir. Dis. 65, 114-124.

9. Metcalf, J. A., Gallin, J. I., Nauseef, W. M., Root, R. K. (1986)Laboratory Manual of Neutrophil Function, Raven Press Ltd., New York.

10. Shephard, E. G., Anderson, R., Rosen, O., Myer, M. S., Fridkin, M.,Strachan, A. F. & De Beer, F. C. (1990) Peptides generated fromC-reactive protein by a neutrophil membrane protease. Amino acidsequence and effects of peptides on neutrophil oxidative metabolism andchemotaxis. J Immunol., 145, 1469-1476.

11. Shephard, E. G., Kelly, S. L., Anderson, R & Fridkin, M. (1992)Characterization of neutrophil-mediated degradation of human C-reactiveprotein and identification of the protease. Chin Exp. Immunol, 87,509-513.

12. Vachino, G., Heck, L. W., Gelfand, J. A., Kaplan, M. M., Burke, J.F., Berninger, R. W., McAdam, K. P. (1988) Inhibition of humanneutrophil and Pseudomonas elastases by the amyloid P-component: Aconstituent of elastic fibers and amyloid deposits. J. Leukocyte Biol.44, 529-534.

13. Yavin, E. J., Rosen, O., Pontet, M., Shephard, E. G., Fridkin, M.(1995) Proteolysis of human C-reactive protein by neutrophil-derivedlysosomal enzymes generates peptides which modulate neutrophil function:Implication to the anti-inflammatory mechanism. Letters in PeptideScience, 2, 7-16.

14. Yavin, E. J., Yan, L., Desiderio, D. M. and Fridkin, M. (1996)Synthetic peptides derived from the sequence of human C-reactive proteininhibit the enzymatic activities of human leukocyte elastase and humanleukocyte cathepsin G. Int. J Peptide Protein Res., 48, 465-476.

15. Yavin, E. J., Eisenstein, M. and Fridkin, M. (1997) Binding pocketson the surface of human leukocyte elastase and human leukocyte cathepsinG. Implications to the design of inhibitors derived from humanC-reactive protein. Biomed. Pep. Prot. Nutcleic acids, In press.

12 1 206 PRT Homo sapiens 1 Glu Thr Asp Met Ser Arg Lys Ala Phe Val PhePro Lys Glu Ser Asp 1 5 10 15 Thr Ser Tyr Val Ser Leu Lys Ala Pro LeuThr Lys Pro Leu Lys Ala 20 25 30 Phe Thr Val Cys Leu His Phe Tyr Thr GluLeu Ser Ser Thr Arg Gly 35 40 45 Tyr Ser Ile Phe Ser Tyr Ala Thr Lys ArgGln Asp Asn Glu Ile Leu 50 55 60 Ile Phe Trp Ser Lys Asp Ile Gly Tyr SerPhe Thr Val Gly Gly Ser 65 70 75 80 Glu Ile Leu Phe Glu Val Pro Glu ValThr Val Ala Pro Val His Ile 85 90 95 Cys Thr Ser Trp Glu Ser Ala Ser GlyIle Val Glu Phe Trp Val Asp 100 105 110 Gly Lys Pro Arg Val Arg Lys SerLeu Lys Lys Gly Tyr Thr Val Gly 115 120 125 Ala Glu Ala Ser Ile Ile LeuGly Gln Glu Gln Asp Ser Phe Gly Gly 130 135 140 Asn Phe Glu Gly Ser GlnSer Leu Val Gly Asp Ile Gly Asn Val Asn 145 150 155 160 Met Trp Asp PheVal Leu Ser Pro Asp Glu Ile Asn Thr Ile Tyr Leu 165 170 175 Gly Gly ProPhe Ser Pro Asn Val Leu Asn Trp Arg Ala Leu Lys Tyr 180 185 190 Glu ValGln Gly Glu Val Phe Thr Lys Pro Gln Leu Trp Pro 195 200 205 2 4 PRTArtificial Sequence synthetic 2 Ala Ala Pro Val 1 3 4 PRT ArtificialSequence synthetic 3 Ala Ala Pro Phe 1 4 15 PRT Artificial Sequencesynthetic 4 Asp Ile Leu Ile Phe Trp Ser Lys Asp Ile Gly Tyr Ser Phe Thr1 5 10 15 5 15 PRT Artificial Sequence synthetic 5 Glu Ile Leu Ile PheTrp Pro Lys Asp Ile Gly Tyr Ser Phe Thr 1 5 10 15 6 15 PRT ArtificialSequence synthetic 6 Glu Ile Leu Ile Phe Trp Ser Xaa Asp Ile Gly Tyr SerPhe Thr 1 5 10 15 7 15 PRT Artificial Sequence synthetic 7 Glu Ile LeuIle Phe Trp Ser Xaa Asp Ile Gly Tyr Ser Phe Thr 1 5 10 15 8 15 PRTArtificial Sequence synthetic 8 Glu Ile Leu Ile Phe Trp Ser Lys Ala IleGly Tyr Ser Phe Thr 1 5 10 15 9 15 PRT Artificial Sequence synthetic 9Glu Ile Leu Ile Phe Trp Ser Lys Glu Ile Gly Tyr Ser Phe Thr 1 5 10 15 1015 PRT Artificial Sequence synthetic 10 Thr Phe Ser Tyr Gly Ile Asp LysSer Trp Phe Ile Leu Ile Glu 1 5 10 15 11 10 PRT Artificial Sequencesynthetic 11 Glu Ile Leu Ile Phe Trp Ser Ala Asp Ile 1 5 10 12 10 PRTArtificial Sequence synthetic 12 Glu Ile Leu Ile Phe Trp Ser Val Asp Ile1 5 10

What is claimed is:
 1. A composition comprising a peptide capable ofinhibiting in vitro the enzymatic activity of human Leukocyte Elastase(hLE) and/or of human Cathepsin G (hCG), said peptide being selectedfrom the group consisting of: (i) a core peptide corresponding topositions 62-71 of the sequence of human C-reactive protein (CRP) of theformula: Glu₆₂-Ile-Leu-Ile-Phe-Trp-Ser-Lys-Asp-Ile₇₁ (residues 62-71 ofSEQ ID NO:1)  or peptides resulting from modification thereofcharacterized by: (ii) substitution of Glu₆₂ by Asp or by a non-aminoacid negatively charged residue derived from succinic, glutaric oradipic acids; (iii) substitution of any of the residues Ile₆₃, Leu₆₄,Ile₆₅, Phe₆₆ or Trp₆₇ by a natural or non-natural hydrophobic amino acidresidue; (iv) deletion of 1 or 2 amino acid residues selected from thegroup consisting of Ile₆₃, Leu₆₄, Ile₆₅, Phe₆₆ and Trp₆₇; (v)substitution of 1-3 amino acid residues selected from the groupconsisting of Ile₆₃, Leu₆₄, Ile₆₅, Phe₆₆ and Trp₆₇ by a singlenon-natural amino acid residue derived from 6-aminocaproic acid; (vi)substitution of 2-4 amino acid residues selected from the groupconsisting of Ile₆₃, Leu₆₄, Ile₆₅, Phe₆₆ and Trp₆₇ by a singlenon-natural amino acid residue derived from 8-aminocaproic acid or10-aminodecanoic acid; (vii) substitution of 3-5 amino acid residuesselected from the group consisting of Ile₆₃, Leu₆₄, Ile₆₅, Phe₆₆ andTrp₆₇ by a stretch of identical hydrophobic amino acid residues or by asingle non-natural amino acid residue derived from 10-aminodecanoic acidor 12-aminolauric acid; (viii) substitution of Ser₆₈ by a natural ornon-natural amino acid residue selected from the group consisting ofThr, Cys, Ala and homoserine; (ix) substitution of Lys₆₉ by a natural ornon-natural positively charged or hydrophobic amino acid residue; (x)substitution of Asp₇₀ by a negatively charged or a polar amino acidresidue selected from the group consisting of Glu, Asn or Gln; (xi)substitution of Ile₇₁ by a natural or non-natural hydrophobic amino acidresidue; (xii) elongation of a peptide (i) to (xi) by 1-5 non-chargedamino acid residues at the N-terminus and/or at the C-terminus; (xiii)substitution of any amino acid residue in a peptide (i) to (xii) by thecorresponding N-alkyl derivative, D-amino acid residue or by anotherisoster; (xiv) an amide of the C-terminal of a peptide (i) to (xiii);and (xv) an N-acyl derivative of a peptide (i) to (xiv).
 2. Thecomposition according to claim 1, wherein the hydrophobic amino acidresidue of (iii) or (xi) is selected from the group consisting of Val,Leu, Ile, Phe, Trp, Tyr, Nva, Nle, homoleucine, homoisoleucine andaminobutyric acid.
 3. The composition according to 1, wherein thepositively charged amino acid residue of (ix) is Arg, His, homolysine,ornithine (Orn) or diaminobutyric acid (DAB).
 4. The compositionaccording to claim 1, wherein the hydrophobic amino acid residue of(vii) or (ix) is Ala, Val, Leu, Ile, Phe, Nva or Nle.
 5. The compositionaccording to claim 1, wherein said peptide is an N-acyl derivative of apeptide (i) to (xiv) and wherein said acyl is a radical R—X—CO—, whereinR is substituted or unsubstituted hydrocarbyl and X is a covalent bond,O, NH, or NHCO.
 6. The composition according to claim 5, wherein saidacyl radical is selected from the group consisting of octanoyl,monomethoxysuccinyl, carbobenzoxy(benzyl-O—CO—), acetylaminocaproyl,Fmoc (fluorenylmethoxycarbonyl), naphthyl-NH—CO— and adamantyl-NH—CO—.7. A composition according to claim 1, wherein said peptide is, selectedfrom the group of sequences consisting of:Glu-Ile-Leu-Ile-Phe-Trp-Ser-Lys-Asp-Ile-Gly-Tyr-Ser-Phe-Thr (pep6)(residues 62-76 of SEQ ID NO:1),Glu-Ile-Leu-Ile-Phe-Trp-Ser-Lys-Asp-Ile-Gly-Tyr (pep13) (residues 62-73of SEQ ID NO:1), Glu-Ile-Leu-Ile-Phe-Trp-Ser-Lys-Asp-Ile (pep14)(residues 62-71 of SEQ ID NO:1),Asp-Ile-Leu-Ile-Phe-Trp-Ser-Lys-Asp-Ile-Gly-Tyr-Ser-Phe-Thr (pep18) (SEQID NO:4), Glu-Ile-Leu-Ile-Phe-Trp-Ser-Orn-Asp-Ile-Gly-Tyr-Ser-Phe-Thr(pep20) (SEQ ID NO:6),Glu-Ile-Leu-Ile-Phe-Trp-Ser-DAB-Asp-Ile-Gly-Tyr-Ser-Phe-Thr (pep21) (SEQID NO:7), Glu-Ile-Leu-Ile-Phe-Trp-Ser-Lys-Ala-Ile-Gly-Tyr-Ser-Phe-Thr(pep22) (SEQ ID NO:8),Glu-Ile-Leu-Ile-Phe-Trp-Ser-Lys-Glu-Ile-Gly-Tyr-Ser-Phe-Thr (pep23) (SEQID NO:9), Glu-Ile-Leu-Ile-Phe-Trp-Ser-Ala-Asp-Ile (pep25) (SEQ IDNO:11), and Glu-Ile-Leu-Ile-Phe-Trp-Ser-Val-Asp-Ile (pep26) (SEQ IDNO:12).
 8. A composition according to claim 1, further comprising apharmaceutically acceptable carrier.
 9. A substantially purified peptidecapable of inhibiting in vitro the enzymatic activity of human LeukocyteElastase (hLE) and/or of human Cathepsin G (hCG), said peptide beingselected from the group consisting of: (i) a core peptide correspondingto positions 62-71 of the sequence of human C-reactive protein (CRP) ofthe formula: Glu₆₂-Ile-Leu-Ile-Phe-Trp-Ser-Lys-Asp-Ile-₇₁ (residues62-71 of SEQ ID NO:1)  or peptides resulting from modification thereofcharacterized by: (ii) substitution of Glu₆₂ by Asp or by a non-aminoacid negatively charged residue derived from succinic, glutaric oradipic acids; (iii) substitution of any of the residues Ile₆₃, Leu₆₄,Ile₆₅, Phe₆₆ or Trp₆₇ by a natural or non-natural hydrophobic amino acidresidue; (iv) deletion of 1 or 2 amino acid residues selected from thegroup consisting of Ile₆₃, Leu₆₄, Ile₆₅, Phe₆₆ and Trp₆₇; (v)substitution of 1-3 amino acid residues selected from the groupconsisting of Ile₆₃, Leu₆₄, Ile₆₅, Phe₆₆ and Trp₆₇ by a singlenon-natural amino acid residue derived from 6-aminocaproic acid; (vi)substitution of 2-4 amino acid residues selected from the groupconsisting of Ile₆₃, Leu₆₄, Ile₆₅, Phe₆₅ and Trp₆₇ by a singlenon-natural amino acid residue derived from 8-aminocaproic acid or10-aminodecanoic acid; (vii) substitution of 3-5 amino acid residuesselected from the group consisting of Ile₆₃, Leu₆₄, Ile₆₅, Phe₆₆ andTrp₆₇ by a stretch of identical hydrophobic amino acid residues or by asingle non-natural amino acid residue derived from 10-aminodecanoic acidor 12-aminolauric acid; (viii) substitution of Ser₆₈ by a natural ornon-natural amino acid residue selected from the group consisting ofThr, Cys, Ala and homoserine; (ix) substitution of Lys₆₉ by a natural ornon-natural positively charged or hydrophobic amino acid residue; (x)substitution of Asp₇₀ by a negatively charged or a polar amino acidresidue selected from the group consisting of Glu, Asn or Gln; (xi)substitution of Ile₇₅ by a natural or non-natural hydrophobic amino acidresidue; (xii) elongation of a peptide (i) to (xi) by 1-5 non-chargedamino acid residues at the N-terminus and/or at the C-terminus; (xiii)substitution of any amino acid residue in a peptide (i) to (xii) by thecorresponding N-alkyl derivative, D-amino acid residue or by anotherisoster; (xiv) an amide of the C-terminal of a peptide (i) to (xiii);and (xv) an N-acyl derivative of a peptide (i) to (xiv).
 10. Thesubstantially purified peptide according to claim 9, wherein thehydrophobic amino acid residue of (iii) or (xi) is selected from thegroup consisting of Val, Leu, Ile, Phe, Trp, Tyr, Nva, Nle, homoleucine,homoisoleucine and aminobutyric acid.
 11. The substantially purifiedpeptide according to 9, wherein the positively charged amino acidresidue of (ix) is Arg, His, homolysine, ornithine (Orn) ordiaminobutyric acid (DAB).
 12. The substantially purified peptideaccording to claim 9, wherein the hydrophobic amino acid residue of(vii) or (ix) is Ala, Val, Leu, Ile, Phe, Nva or Nle.
 13. Thesubstantially purified peptide according to claim 9, which is an N-acylderivative of a peptide (i) to (xiv) and wherein said acyl is a radicalR—X—CO—, wherein R is substituted or unsubstituted hydrocarbyl and X isa covalent bond, O, NH, or NHCO.
 14. The substantially purified peptideaccording to claim 13, wherein said acyl radical is selected from thegroup consisting of octanoyl, monomethoxysuccinyl,carbobenzoxy(benzyl-O—CO—), acetylaminocaproyl, Fmoc(fluorenylmethoxycarbonyl), naphthyl-NH—CO— and adamantyl-NH—CO—.
 15. Asubstantially purified peptide according to claim 9, selected from thegroup of sequences consisting of:Glu-Ile-Leu-Ile-Phe-Trp-Ser-Lys-Asp-Ile-Gly-Tyr-Ser-Phe-Thr (pep6)(residues 62-76 of SEQ ID NO:1),Glu-Ile-Leu-Ile-Phe-Trp-Ser-Lys-Asp-Ile-Gly-Tyr (pep13) (residues 62-73of SEQ ID NO:1), Glu-Ile-Leu-Ile-Phe-Trp-Ser-Lys-Asp-Ile (pep14)(residues 62-71 of SEQ ID NO:1),Asp-Ile-Leu-Ile-Phe-Trp-Ser-Lys-Asp-Ile-Gly-Tyr-Ser-Phe-Thr (pep18) (SEQID NO:4), Glu-Ile-Leu-Ile-Phe-Trp-Ser-Orn-Asp-Ile-Gly-Tyr-Ser-Phe-Thr(pep20) (SEQ ID NO:6),Glu-Ile-Leu-Ile-Phe-Trp-Ser-DAB-Asp-Ile-Gly-Tyr-Ser-Phe-Thr (pep21) (SEQID NO:7), Glu-Ile-Leu-Ile-Phe-Trp-Ser-Lys-Ala-Ile-Gly-Tyr-Ser-Phe-Thr(pep22) (SEQ ID NO:8),Glu-Ile-Leu-Ile-Phe-Trp-Ser-Lys-Glu-Ile-Gly-Tyr-Ser-Phe-Thr (pep23) (SEQID NO:9), Glu-Ile-Leu-Ile-Phe-Trp-Ser-Ala-Asp-Ile (pep25) (SEQ IDNO:11), and Glu-Ile-Leu-Ile-Phe-Trp-Ser-Val-Asp-Ile (pep26) (SEQ IDNO:12).